Two major problems associated with the administration of drugs have been poor pharmacokinetics (PK) and undesired drug-drug interactions. It is known that the cytochrome P450 (CYP) 3A family of enzymes are important in human drug metabolism because they metabolize the majority of commercially available drugs (Wrighton et al., Drug metabolism reviews 32: 339-361 (2000)). In 1998, the pregnane X receptor (PXR), which orchestrates the induction of some of the major genes involved in drug detoxification, was identified. (Lehmann, et al. J. Clin. Invest. 102(5):1016-1023 (1998); Genbank Acccession No. AF061056). PXR, also known as steroid and xenobiotic receptor (SXR or PAR), was shown to be activated by many drugs, steroids such as pregnenolone 16α-carbonitrile; RU-486; dexamethasone; and rifampicin (Kliewer et al., Cell 92(1):73-82 (1998); Kliewer et al., J. Lipid Res. 43:359-364 (2002)) and St. John's wort (hyperforin) (Moore et al., Proc. Natl. Acad. Sci. USA.; 97(13):7500-7502 (2000)) and recently was shown to be a sensor for bile acids (Staudinger et al., Drug Metab. Dispos. 29(11):1467-1472 (2001); Staudinger et al., Proc. Natl. Acad. Sci. USA. 98(6):3369-74 (2001)). PXR is also activated by other drugs such as clotrimazole (Lehmann et al., J. Clin. Invest. 102:1016-1023 (1998)); 11β-hydroxylase inhibitor metyrapone (Goodwin et al., Mol. Pharmacol. 56:1329-1339 (1999); Wright et al., Biochem. Soc. Trans. 27:387-391 (1998)); troglitazone (Jones et al, Mol. Endocrinol. 14:27-39 (2000)); ritonavir (Dussault et al., J. Biol. Chem. 276:33309-33312 (2001)) and taxol (Synold et al., Nat. Med. 7:584-590 (2001)) as well as by environmental pollutants such as bisphenol A, diethylhexylphthalate, and nonylphenol (Masuyama et al., Mol. Endocrinol. 14:421-428 (2000); Takeshita et al., Eur. J. Endocrinol. 145:513-517 (2001)). PXR plays a key role in the regulation of both drug metabolism and efflux by modulating a plethora of genes encoding cytochrome P450 enzymes (CYPs, especially CYP3A4) and a multidrug resistant gene ABCB1 (Synold et al., Nat. Med. 7(5):584-590 (2001)). Together, these proteins are responsible for the elimination of >50% of all drugs. Identification of candidate pharmaceutical treatments which interact with PXR and are likely to be metabolized by the PXR system is enormously useful information when performing an early evaluation of the safety and pharmacokinetic profile of the treatment. Therefore, high-throughput screening assays detecting PXR-mediated induction have become pivotal at early discovery stages in order to decrease the time line for clinical drug development. Moreover, crystals of PXR are useful for these purposes in that they provide a greater understanding of ligand interactions with the protein and allow computer-assisted, structure-based evaluation of candidate treatments.
Several crystals comprising PXR are known in the art. For example, the structure of apo-PXR-LBD crystal comprising space group P43212 was solved with a 2.52 Å resolution (Watkins et al., Science 292:2329-2333 (2001)); the structure of PXR-LBD/SR12813 crystal comprising space group P43212 was solved with a 2.76 Å resolution (Watkins et al., Science 292:2329-2333 (2001)); the structure of PXR-LBD/Hyperforin comprising space group P43212 was solved with a 2.15 Å resolution (Watkins et al., Biochemistry 42:1430-1438 (2003)).
In addition, the structure of PXR-LBD/SR12813/SRC1 crystal comprising space group P212121 was solved with a resolution of 2.00 Å (Watkins et al., J. Mol. Biol. 331:815-828 (2003)). The crystal in Watkins et al., however, comprised PXR-LBD complexed with SRC1 and not covalently bound in a hybrid.